Oxidative stress significantly contributes to the progression of diabetes. The use of antioxidants or α-glycosidase inhibitors would be an effective therapeutic strategy for diabetes. To discover natural antioxidants or α-glycosidase inhibitors, two new compounds, penidione A (1) and peniorcinol D (2), were obtained from the endophytic fungus Penicillium brefeldianum F4a isolated from the roots of Houttuynia cordata Thunb. (Saururaceae). The structures were identified through a comprehensive analysis of spectroscopic data, including HR-ESI-MS, UV, IR, 1D and 2D NMR, together with quantum chemical calculations. Biological tests indicated that penidione A displayed weaker α-glycosidase inhibition activity than that of acarbose and peniorcinol D showed significant ABTS